This project focuses on the evolutionary molecular genetics of the mammalian beta-globin gene clusters in which gamma genes occur. An important aim is to develop phylogenetic footprinting procedures which when applied to sufficient bodies of comparative sequence data can distinguish within stretches of non-coding sequence the evolutionarily conserved elements from neutral DNA. Achieving this aim will facilitate accomplishing the project's major objective: namely, to elucidate the genetic program that evolved in humans and other simian primates for switching from embryonic (zeta2 sigma2; alpha2 sigma2) to fetal (alpha2 gamma2) to adult (alpha2 delta2; alpha2 beta2) hemoglobin synthesis. The differing developmental pattern found in mice, rabbits, and the prosimian primate galago (embryonic but not fetal expression of gamma-globin genes and the full switch form gamma to beta expression in fetal life rather than after birth) indicates that phylogenetic footprinting will help identify cis-acting DNA sequences involved in the developmental switching from sigma to gamma to delta and beta expression in the human beta-globin gene cluster. The genetic program for hemoglobin switching that evolved in primates provides a model for any developmentally controlled human gene system in which mutations in regulatory elements cause clinical disorders. The DNA sequencing now in progress on beta-globin gene clusters from galago, tarsier (the prosimian closest to simians), spider monkey, rhesus monkey, gibbon, oragnutan, gorilla, and chimpanzee will be extended to include not only most genes within these clusters but the majority of flanking and intergenic regions. Extensive DNA sequencing of the beta-globin gene clusters of additional simian and prosimian species and of several non-primate placental mammals (flying lemur, tree shrew, and armadillo) is also planned. Phylogenetic footprinting of this enlarge body of sequenced data, in helping to establish whether the fetal program of gamma expression evolved on the stem of Haplorhini (Tarsier and simians) or in the succeeding stem of Anthropoidea, could identify adaptive sequence elements that after emergence in the pre-simians were conserved in simian gene clusters. Complementary studies using erythroid cell culture systems will test expression of gamma-containing DNA clones from prosimians which embryonic gamma programs (e.g. galago) and form simians with their fetal gamma program in order to further assess the respective roles of trans-factors and cis- elements in the evolutionary recruitment of gamma to a fetal development program.